Progressive deterioration of myocardial function during acute and/or chronic heart disease probably involves defects in Ca2+ uptake and release by the cardiac sarcoplasmic reticulum (SR). This study will examine and elucidate the role of cyclic AMP (cAMP) dependent and Ca2+-calmodulin dependent phosphorylations of cardiac SR in regulating its energy-dependent Ca2+ transport activity. Cardiac SR will be phosphorylated to various degrees by cAMP-dependent and by Ca2+-calmodulin dependent protein kinases. The individual and the combined effects of these phosphorylations on Ca2+ transport and Ca2+ ATPase activity will be examined under steady-state and presteady-state conditions. Using a specially fabricated chemical quench-flow apparatus, the individual steps in the Ca2+ ATPase reaction sequence which are regulated by either cAMP-dependent or Ca2+-calmodulin dependent phosphorylations will be identified. Alterations in any of the cardiac SR parameters which are controlled by phosphorylation will be correlated with the degree of cardiac SR phosphorylation. Special emphasis will be placed on the cAMP-dependent and Ca2+-calmodulin dependent protein kinases associated with cardiac SR in terms of purification and characterization since these are likely the enzymes involved in regulating SR function in vivo. The proposed research should provide information concerning cAMP-dependent and Ca2+-calmodulin dependent phosphorylation of sarcoplasmic reticulum as regulatory mechanisms for the calcium pump and, therefore, important mechanisms of the contraction-relaxation cycle in cardiac muscle. These basic studies will lead to more rational experiments designed to elucidate the role of SR phosphorylation in models of ischemic heart disease and congestive heart failure.